The authors acknowledge funding from NINDS/NIH, R00-NS064197 and NINDS/NIH, R01NS083897 (both to D.L.S.).

1. Preparation of Media (Figure 1, Step 1)
<ol>
	<li>Slice Culture Medium
	<ol>
		<li>25 ml of slice culture medium is sufficient to prepare 5 glass bottom dishes with 2 slices per well.</li>
		<li>In a 50 ml conical tube, add 250 &#181;l of a 100x N2 solution and 500 &#181;l of a 50x B27 solution without vitamin A. Add DMEM/F12 to a volume of 22.5 ml.</li>
		<li>Filter sterilize solution and subsequently add 1.25 ml of heat inactivated horse serum and 1.25 ml of fetal bovine serum.</li>
		<li>Incubate solution in a water bath at 37&#160;&#176;C&#160;for the duration of the preparation of the slices.</li>
		<li>Add growth factors (FGF and EGF, final concentration of 10 ng/ml and 20 ng/ml, respectively) immediately prior to the beginning of culture. The composition of this medium has been optimized to promote the survival of cells in culture and to increase the proliferation rate of neural progenitors. This will thus maximize the probability to observe mitotic cells in the slice.</li>
	</ol>
	</li>
	<li>Full HBSS Dissection Solution
	<ol>
		<li>Prepare 500 ml of full HBSS by adding 50 ml of 10x HBSS, 1.25 ml of 1 M Hepes (pH 7.4, FC 2.5 mM), 6 ml of 2.5 M D-Glucose (FC 30 mM), 2.2 ml of 0.9 M NaHCO3 (FC 4 mM) and autoclaved diH2O to final volume of 500 ml.</li>
		<li>Filter sterilize solution and store at 4 &#176;C until the collection of uterine horns from the pregnant mouse. This solution can be kept at 4 &#176;C for weeks.</li>
		<li>Keep HBSS on ice during the whole dissection procedure.</li>
	</ol>
	</li>
	<li>Embedding solution
	<ol>
		<li>For the embedding of 4 embryonic brains, prepare 30 ml of 3% low melting agarose in the full HBSS solution. In a 50 ml conical tube, add 0.9 g of low melting agarose and full HBSS to 30 ml.</li>
		<li>Mix the solution with a vortex mixer, and melt in a microwave with the tube standing and the cap open.</li>
		<li>While in the microwave, monitor the solution to prevent overflow due to excessive boiling. When boiling starts, stop the microwave and vortex the solution.</li>
		<li>Repeat these steps until all the agarose has melted.</li>
		<li>Store the tube in a water bath at 42 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Dissection of the Embryos (Figure 1, Step 2)
<ol>
	<li>After careful euthanasia of the pregnant mouse, collect the uterine horns and transfer them into a 10 cm2&#160;Petri dish containing cold full 1x HBSS. The age of the embryo can vary depending upon the study. This protocol has been used successfully for culturing brain slices from embryonic days E12.5 to E17.5. Due to their size, younger brains tend to be more difficult to work with.</li>
	<li>Collect the embryos and dissect out the embryonic brains as described previously in the rat and mouse26,27, until a full embryonic brain including the hindbrain and the forebrain with the two&#160;cerebral hemispheres is obtained. These can be kept at RT until all brains have been dissected.</li>
	<li>Collect the embryos and dissect out the embryonic brains as described previously in the rat and mouse26,27, until a full embryonic brain including the hindbrain and the forebrain with the two&#160;cerebral hemispheres is obtained. These can be kept at RT until all brains have been dissected.</li>
</ol>
3. Embedding of the Embryonic Brains (Figure 1, Step 3)
<ol>
	<li>In a bucket containing ice, create a hole in ice to insert the embedding molds into.</li>
	<li>Pour the 3% agarose medium into a plastic mold and place that mold into the hole prepared in the ice.</li>
	<li>Stir the agarose medium with the tip of a digital thermometer until the temperature reaches 35 &#176;C.</li>
	<li>Promptly transfer the brain into the embedding medium.</li>
	<li>Critical Step: To remove the excess HBSS at the interface between the brain and the agarose, carefully and gently tumble/rotate the brain repeatedly in the embedding solution with the forceps.</li>
	<li>A cushion of gelled agarose will form at the bottom of the mold in contact with the ice. Once the cushion can be felt with the tip of the forceps while stirring the brain, position the brain with the dorsal side up. The brain should not sink to the very bottom of the mold.</li>
</ol>
4. Preparation of Agarose Block (Figure 1, Step 4)
<ol>
	<li>Let the agarose harden in the ice for at least 5 min.</li>
	<li>Using a razor blade, section the corners of the mold.</li>
	<li>Carefully carve an agarose block around the brain using a razor blade. Try to minimize the number of cuts to avoid perturbing the embedded brain.</li>
	<li>Make sure that the block has a larger surface area in the caudal region of the brain (versus the rostral region, see Figure 1, Step 4) to increase the stability of the block on the sectioning pedestal.</li>
	<li>The last cut should be the one on the caudal side of brain; this cut will define the correct sectioning plane with the vibratome.</li>
	<li>Do so by aligning the blade perpendicular to the rostro-caudal axis of the brain and the agarose block. Slide down the blade caudally along the rostro-caudal axis and make the final cut about 5 mm away from the most caudal region of the brain.</li>
	<li>Set the agarose/brain block aside and repeat embedding of other brains.</li>
</ol>
5. Transfer of the Embedded Brains into the Tray of the Vibratome (Figure 1, Step 5)
<ol>
	<li>Add a drop of glue onto the bottom of the vibratome tray. Place glue so that the agarose blocks will be positioned within reach of the vibrating blade.</li>
	<li>Place the agarose/brain block caudal side down on the edge of the spatula, with about 50% of the block tipping over the edge of the spatula.</li>
	<li>Apply the caudal face of the agarose block to the glue and slide the spatula away, maintaining the agarose block to the bottom of the tray with the shoulder of a pair of tweezers. Do not let the spatula directly contact the glue.</li>
	<li>Let the glue solidify at room temperature for 5 min.</li>
</ol>
6. Sectioning of the Embedded Brains (Figure 1, Step 6)
<ol>
	<li>Fill the vibratome tray with HBSS.</li>
	<li>Define the start and finish positions of the vibratome blade.</li>
	<li>Generate 200 - 250 &#181;m slices. With the VT1000s vibratome, use the following parameters: speed 2 - 4, frequency 8.</li>
	<li>Carefully remove the slices from the vibratome as they are cut. Transfer the slices with a spatula and the back end of tweezers into 12 well dishes filled with full HBSS. Alternatively some researchers have used a paintbrush in lieu of tweezers. Key point: During transfer, take care that the brain slices remain attached to the surrounding agarose.</li>
</ol>
7. Syto11 Staining of the Slices (Figure 1, Step 7)
<ol>
	<li>Dilute Syto11 to a final concentration of 0.5 - 1 &#181;M in the slice culture medium supplemented with growth factors.</li>
	<li>In the well of a 12-well plate, incubate slices from one brain in 2.5 ml of the staining solution for 1 hr at 37 &#176;C.</li>
	<li>Wash the slices in 2.5 ml of slice culture medium without staining solution for 20 min.</li>
</ol>
8. Mounting the Slices in a Glass Bottom Dish (Figure 1, Steps 8, 9)
<ol>
	<li>Prepare 15 &#181;l of embedding collagen solution per slice. Prepare a 1.5 mg/ml collagen solution by mixing 375 &#181;l of 3 mg/ml collagen type I solution with 75 &#181;l of 10x DMEM, 9.4 &#181;l of 1 M NaOH, and 290 &#181;l of H2O. Store on ice.</li>
	<li>Place a 15 &#181;l drop of the collagen solution at the bottom of a 35 mm Glass bottom Microwell Dish. Spread it with a pipet tip to match the size of the slice.</li>
	<li>Transfer the slice into the drop of collagen with a spatula and pair of tweezers. Key point: Mounting multiple slices in different dishes increases the probability to acquire slices suitable for imaging. Screen through different slices and select the ones that best meet the criteria described below in the &#34;Representative Results&#34; section.</li>
	<li>Let the slices incubate at RT for 10 min before transferring them into a 37 &#176;C incubator with 5% CO2.</li>
	<li>After 20 min, add 1.2 ml of slice culture medium into the Glass Bottom Microwell Dish. Add 600 &#181;l of medium at a time and spread it with a pipette tip.</li>
	<li>Capture a low magnification image of the brain slice to record the anatomical level and integrity of the slice.</li>
</ol>
9. Live Imaging of the Slices (Figure 1, Steps 10, 11)
<ol>
	<li>Let the slices recover in the incubator for at least 1 hr and 30 min at 37 &#176;C with 5% CO2 before live imaging.</li>
	<li>Image the slices with the microscope of choice, keeping in mind that Syto11 is very prone to photobleaching. Here an inverted spinning disk confocal microscope is used (Andor XD revolution spinning disk confocal microscope). Alternatively a laser scanning confocal microscope can be used. The following parameters are for the spinning disk microscope set-up.</li>
	<li>During the entire live imaging session, maintain slices at 37 &#176;C, 5% CO2 in a humidified incubation chamber attached to the microscope.</li>
	<li>Image cells with a 60X silicon oil objective with a working distance of 300 &#181;m, and a numerical aperture of 1.3 (for examples see Figures 3B, 3C, 3E, 3F, 4A, 4B). A 100X oil objective with a working distance of 130 &#181;m and a numerical aperture of 1.4 (for example see Figure 4C) can also be used. The resolution of the camera is 512 x 512.</li>
	<li>Image the cells in a 30 &#181;m z-stack, with the center of the z-stack located about 40 &#181;m below the surface of the slice. Trying to image deeper into the tissue can cause the objective to add mechanical pressure on the slice. This can affect the integrity of the brain slice. If planning to make 3D reconstructions of the cells, use a z-interval of no more than 2 &#181;m.</li>
	<li>Adjust the laser power and exposure times to limit photobleaching. Use exposure times ranging from 30 to 200 msec, depending on the intensity of the Syto11 signal.</li>
	<li>With a motorized stage, image multiple positions across multiple slices mounted in 1 dish. For example, image up to 20 positions scattered across 4 different slices in 1 single dish.</li>
	<li>For live imaging, use a temporal resolution of less than 5 min to enable the identification of the different phases of mitosis. Using Syto11 and/or histone H2B-EGFP, 4 - 5 hr of live imaging are sufficient to observe a significant number of mitotic cells. However, if imaging sparsely labeled mitotic cells (such as those achieved by in utero electroporation), then a longer imaging parameter (overnight) is appropriate and works well. 
	Note: Using this protocol, we typically observe on average 10 mitotic cells per position. As stated above, imaging multiple mounted slices increases the probability of having a successful experiment. With this approach we typically identify mitotic cells in virtually all experiments performed with Syto11 or histone H2B-EGFP.</li>
</ol>
10. Post-acquisition Analysis of Mitosis (Figure 2)
All the procedures in this section have been optimized in Fiji (ImageJ), which is advantageous because it is a free open source software. Other software solutions are available to perform the same tasks, such as Metamorph, Imaris, and Amira.
<ol>
	<li>Identification of Mitotic Figures in Fiji
	<ol>
		<li>After opening the dataset for one position as a &#34;hyperstack&#34;, select one z-plane (see Figure 2A for the location of the scroll bar) where the tissue and cells look healthy (see criteria in the discussion section). Scroll across the temporal dimension to identify cells going through anaphase, as it is the easiest mitotic phase to identify.</li>
		<li>Scroll back in time to identify the time point where the cell enters mitosis (DNA condensation). The cell may move across z-planes, but the temporal resolution and the z-stack parameters described earlier have been optimized to enable this process.</li>
		<li>In a spreadsheet, record the 4D coordinates of the cell in the hyperstack (X,Y,Z and time, see Figure 2A to visualize the location of these figures in the Fiji interface) as it enters mitosis. Knowing these coordinates will prevent counting the same cell several times.</li>
		<li>Scroll forward in time and record the time when the cell progresses from one phase to another. Document the duration of each mitotic phase for one given cell.</li>
		<li>Proceed with other cells, across the full dataset (multiple cells within and across positions).</li>
	</ol>
	</li>
	<li>3D Reconstruction of the Cells and Quantitation of Rotation During Metaphase (Adapted from Haydar et al, 2003) (Figure 2B).
	<ol>
		<li>After having identified multiple mitotic cells, use the coordinates of a cell and scroll forward in time until the beginning of metaphase.</li>
		<li>Rotate the entire &#34;hyperstack&#34; so that the ventricular border is planar (using the &#34;Image/Transform/Rotate&#8230;&#34; command). Use the &#34;angle&#34; tool to determine the angle to apply for this rotation.</li>
		<li>Identify the z-plane where the cell of interest is the most visible. In that plane, use the &#34;rectangle selection&#34; tool to draw a selection that includes the whole cell.</li>
		<li>Identify the z-planes that include the cell of interest. Use the &#34;Image/Duplicate&#34; command to create a &#34;sub-stack&#34;. In the dialog box, leave &#34;Duplicate hyperstack&#34; checked. The &#34;Slices (z)&#34; parameter corresponds to the z-planes including the whole cell, and the &#34;Frames (t)&#34; parameter corresponds to the current time point.</li>
		<li>If the resolution is poor, increase the size of the &#34;sub-stack&#34; using the &#34;Image/Adjust/Size&#8230;&#34; command.</li>
		<li>Generate a 3D reconstruction of the cell at the current time point using the &#34;Image/Stacks/3D project&#8230;&#34; command. The parameters for this command are displayed in Figure 2B. The &#34;Slice spacing&#34; corresponds to the interval between each of the z-plane images (&#181;m). Important: make sure that the physical dimension (&#181;m) of the sub-stack has been conserved. If not, the interpolation of the pixels in the z dimension will be incorrect, and the 3D reconstruction will be inaccurate.</li>
		<li>Using the scrollbar, rotate the resulting 3D reconstruction so that the edge of the metaphase plate is visible. The number of the frame corresponds to the beta angle described in Figure 3B, record this number. Using the &#34;angle&#34; tool and the &#34;Analyze/Measure&#8230;&#34; command, measure the angle between the horizontal and a line bisecting the metaphase plate perpendicularly (Figure 2B). This is the angle alpha.</li>
		<li>Record these angles for all the metaphase time points of the cell of interest. The angle of rotation between time points (t) and (t-1) equals to the absolute value of the difference between an angle at (t) and this angle at (t-1).</li>
	</ol>
	</li>
	<li>Measuring the Orientation of the Cleavage Plane
	<ol>
		<li>3D reconstruct a cell of interest at one time point during anaphase following the instructions described for the 3D reconstruction of metaphase plates.</li>
		<li>Rotate the reconstruction until the edges of the two plates formed by the segregating chromosomes are visible.</li>
		<li>Use the angle tool to measure the angle between the horizontal (the ventricular edge) and a line parallel to the two plates formed by the segregating chromosomes. This is the angle of the cleavage plane (Figure 2C).</li>
	</ol>
	</li>
	<li>Generation of Movies
	<ol>
		<li>As the cells often move across different z-planes, identify the z-planes occupied by the cell during the live imaging session. Generate a &#34;Maximum intensity projection&#34; of these z-planes using the &#34;Image/Stacks/Z project&#8230;&#34; command. Save the resulting stack as a &#34;.TIF&#34; file.</li>
		<li>Open this file in the 1.45 version of ImageJ as Fiji does not include a stable plugin for the generation of &#34;.mov&#34; or &#34;.avi&#34; files.</li>
		<li>Use the &#34;File/Save as/&#8230;&#34; command of ImageJ to generate a movie in a format compatible with your down-stream applications.</li>
	</ol>
	</li>
</ol>

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C., Mart&#237;nez-Cerde&#241;o, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nature Publishing Group. 7, 136-144 (2004).</li><li>Tyler, W. A., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genetic+Fate+Mapping+Reveals+a+Novel+Route+of+Neocortical+Neurogenesis,+Which+Is+Altered+in+the+Ts65Dn+Mouse+Model+of+Down+Syndrome.">Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome.</a> Journal of Neuroscience. 33, 5106-5119 (2013).</li><li>Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-Lapse+Imaging+of+Fluorescently+Labeled+Live+Cells+in+the+Embryonic+Mammalian+Forebrain.">Time-Lapse Imaging of Fluorescently Labeled Live Cells in the Embryonic Mammalian Forebrain.</a> CSH Protoc. , (2011).</li><li>Elias, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+of+E18+rat+brains.">Organotypic slice culture of E18 rat brains.</a> Journal of visualized experiments : JoVE. , (2007).</li><li>Shitamukai, A., Konno, D., Matsuzaki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oblique+radial+glial+divisions+in+the+developing+mouse+neocortex+induce+self-renewing+progenitors+outside+the+germinal+zone+that+resemble+primate+outer+subventricular+zone+progenitors.">Oblique radial glial divisions in the developing mouse neocortex induce self-renewing progenitors outside the germinal zone that resemble primate outer subventricular zone progenitors.</a> Journal of Neuroscience. 31, 3683-3695 (2011).</li><li>Hadjantonakis, A. -. K., Papaioannou, V. 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L., Van Aelst, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DOCK7+interacts+with+TACC3+to+regulate+interkinetic+nuclear+migration+and+cortical+neurogenesis.">DOCK7 interacts with TACC3 to regulate interkinetic nuclear migration and cortical neurogenesis.</a> Nat Neurosci. 15, (2012).</li><li>Siegenthaler, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+Acid+from+the+meninges+regulates+cortical+neuron+generation.">Retinoic Acid from the meninges regulates cortical neuron generation.</a> Cell. 139, 597-609 (2009).</li><li>Schenk, J., Wilsch-Br&#228;uninger, M., Calegari, F., Huttner, W. 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The authors declare they have no competing financial interests.

The major advantage of the protocol we have described is that it provides a dynamic temporal resolution of mitosis of neural progenitors. Typically, assays to visualize mitosis in the developing brain are performed using immunofluorescence of fixed tissue sections. But this approach only provides a snapshot of mitosis at one time point.
There are several steps that are most critical for imaging mitosis in brain slices: 1) The brain slices should remain attached to the agarose, to preserve the ...

The overall goal of this protocol is to describe how to perform live imaging of neural progenitor mitosis in embryonic brain slices. Using live imaging of brain slices in culture, this protocol provides a simple method to assay multiple aspects of mitosis in neural progenitors in an environment highly similar to an in vivo setting. It can be applied to brains of mutant animals and/or brains that have been manipulated with in utero electroporation)1-5. This technique is also ideal to test the effect of pharmacological agents on neural precursors&#39; mitosis, by simply adding an agent to the culture medium. In sum, this article will make a technically challenging protocol accessible to those studying neurogenesis.
During neurogenesis, distinct neural progenitor populations undergo precise divisions to generate neurons that eventually contribute to the six cortical layers of the adult neocortex6-8. Early in cortical development, the neural precursor pool expands as neuroepithelial (NE) cells divide symmetrically to self-renew. NE cells then convert into radial glial cells (RGCs). Initially RGCs divide symmetrically to produce two new RGCs, however during the bulk of neurogenesis, RGCs&#39; main mode of division is asymmetric. In asymmetric division, 1 RGC gives rise to a new RGC and either a post-mitotic neuron, or a more specialized progenitor (either a short neural precursor (SNP), an outer radial glia (ORG), or an intermediate progenitor (INP)2,3,7,9. INPs, SNPs, and ORGs can then generate neurons at the sub-ventricular, ventricular, and basal regions of the cortex, respectively. Hence, cell division of progenitors is a fundamental process for generating neurons of the neocortex.
Numerous studies point to a correlation between specific mitotic traits of RGCs and the fate of daughter cells. Haydar et al. and Takahashi et al. have shown that RGC mitotic duration and cell cycle length increase as neurogenesis proceeds, a finding echoed in follow up studies10-13. A number of studies have suggested that mitotic spindle orientation relative to the ventricle influences aspects of neurogenesis and corticogenesis, including types of neurons generated and location of progeny in the brain, respectively3,10,14-16. Whether cleavage plane orientation directly influences cell fate is controversial, but the conclusion remains that this mitotic parameter impacts neurogenesis. Further underscoring the importance of mitosis is the observation that many genes involved in the mechanics of mitosis are crucial for neurogenesis and for proper brain development17-20.
Mitosis is a dynamic process, yet to date most studies detailing neural progenitor mitosis utilize analysis of fixed tissue sections or imaging of neural progenitors via in vitro cell culture. Thus, the mainstream methods to evaluate mitosis only provide a snapshot of this process and fail to uncover how cells behave in a tissue. Live imaging of neural progenitor mitosis has increasingly become a critical tool for understanding neural progenitor function. For examples please see these references4,8,10,21-25. Several outstanding protocols have been published for preparation and imaging of brain slices26,27. However to date, a comprehensive protocol for imaging and analysis of mitosis has not been described nor demonstrated in video.
This technique offers several significant advantages over fixed analysis of brain sections. Time-lapse analysis of brain slices enables generation of significantly more data points that can be analyzed in a flexible fashion. First, data is gathered at individual time points over the course of several minutes or several hours. One can analyze individual time points (to create a static montage) or can combine different time points into movies. Second, confocal imaging of slices enables generation of data at different Z sections in brain slices. As a result, individual sections can be analyzed. Alternatively, stacks of individual sections can be combined into a maximum intensity projection. Third, analysis is done in the context of a tissue, revealing how cells divide relative to neighboring cells and structures. Fourth, it is ideally suited to analysis of mutants that show some evidence of mitotic defects. Together this protocol will help clarify critical steps to aid investigators who wish to carry out live imaging of neural progenitor mitosis in their own laboratories.

<table><tbody><tr><td>100X N2</td><td>Life technologies</td><td>17502048</td><td>for culture medium</td></tr><tr><td>50X B27 without vitamin A</td><td>Life technologies</td><td>12587010</td><td>for culture medium</td></tr><tr><td>DMEM/F12</td><td>Life technologies</td><td>11320033</td><td>for culture medium</td></tr><tr><td>Heat-inactivated horse serum</td><td>Sigma Aldrich</td><td>H1138-500ml</td><td>for culture medium</td></tr><tr><td>Heat-inactivated calf serum</td><td>Sigma Aldrich</td><td>F4135-500ML</td><td>for culture medium</td></tr><tr><td>FGF</td><td>R&amp;amp;D Sytems</td><td>3139-FB-025</td><td>for culture medium</td></tr><tr><td>EGF</td><td></td><td></td><td>for culture medium</td></tr><tr><td>10X HBSS</td><td>Life technologies</td><td>14065-056</td><td>for the dissection of the embryos</td></tr><tr><td>Hepes Free Acid</td><td>Sigma Aldrich</td><td>H4034</td><td>dilute to 1 M (pH 7.4)</td></tr><tr><td>2.5 M D-Glucose</td><td>Sigma Aldrich</td><td>G8769</td><td>for the dissection of the embryos</td></tr><tr><td>0.9 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Life technologies</td><td>25080-094</td><td>for the dissection of the embryos</td></tr><tr><td>Low-melting agarose</td><td>Fisher</td><td>BP165-25</td><td>for generating slices</td></tr><tr><td>Syto11</td><td>Life technologies</td><td>S7573</td><td>Make 5&amp;micro;l aliquots</td></tr><tr><td>3 mg/ml collagen type I</td><td>Life technologies</td><td>A1048301</td><td>for culturing slices</td></tr><tr><td>Glass bottom dish</td><td>MatTek</td><td>P35G-1.5-14-C</td><td>for culturing slices</td></tr><tr><td>Petri dishes</td><td></td><td></td><td>for dissection of the embryos</td></tr><tr><td>Digital thermometer</td><td></td><td></td><td>to measure the temperature of the agarose</td></tr><tr><td>Spatula</td><td></td><td></td><td>to transfer brains</td></tr><tr><td>Paintbrush</td><td></td><td></td><td>alternative to transfer brains</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000s</td><td>for generating slices</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>dissecting out embryos</td></tr><tr><td>Imaging microscope</td><td></td><td></td><td>A confocal microscope is required, it needs to be equipped with an incubation chamber</td></tr></tbody></table>

live imaging of mitosis in the developing mouse embryonic cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

The success of this assay and the observation of multiple mitotic cells during one live imaging session largely depend upon both the integrity and anatomical level of the slice where acquisitions are made. As discussed below, the anatomical level of a slice is an important factor. Figure 3A shows rostro-caudal and medial-lateral locations where we have been most successful. For additional discussion of this subject please see Noctor26. The integrity of the slice can vary in different regions o...

Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

live-imaging-of-mitosis-in-the-developing-mouse-embryonic-cortex

Research

JoVE Journal

Neuroscience

15.2K Views.  Duke University Medical Center. Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Video: Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Developmental Biology

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Biology

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Live Cell Imaging of Mitosis

Mitosis is a form of cell division in which a cell&#8217;s genetic material is divided equally between two daughter cells. Mitosis can be broken down into six phases, during each of which the cell&#8217;s components, such as its chromosomes, show visually distinct characteristics. Advances in fluorescence live cell imaging have allowed scientists to study this process in great detail, providing important insights into the biological control of this process and how it might go wrong in diseases such as cancer. We begin this video by breaking down the phases of mitosis, and introducing some important considerations for optimal visualization of the process using live cell imaging. We then walk through the steps for running a live cell mitosis imaging experiment and discuss various analysis methods, including the generation of montages, movies, and 3D recreations. Finally, we take a look at how visualizing the mitotic process can be applied to answering questions in cell biology.

Mitosis is a form of cell division in which a cell&#8217;s genetic material is divided equally between two daughter cells. Mitosis can be broken down into ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Summary

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Chapters in this video

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Live Cell Imaging of Chromosome Segregation During Mitosis

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Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

Live Cell Imaging of Mitosis